Upconversion-Linked Immunosorbent Assay for the Biomimetic Detection of the Mycotoxin Cyclopiazonic Acid

The neurotoxin α-cyclopiazonic acid (CPA) is an emerging mycotoxin produced as a secondary metabolite by several fungi species (i.e., Penicillium spp. and Aspergillus spp.). CPA commonly contaminates maize, crops, cheese, and wine. In this work, CPA detection in foodstuff was accomplished by the innovative integration of two strategies: upconversion nanoparticles (UCNPs) and epitope-mimicking peptides, to develop a competitive upconversion-linked immunosorbent assay (ULISA). We have applied UCNPs (type NaYF₄:Yb³⁺, Er³⁺) as background-free optical labels due to their anti-Stokes shift with excitation in the near-infrared region and emission in the ultraviolet-visible region. Moreover, a CPA epitope-mimicking cyclic peptide (A2) was used as a substitute for the toxin-conjugates traditionally applied to competitive assays. UCNPs were decorated with an anti-CPA fragment antigen-binding antibody (UCNP-Fab), and CPA detection was achieved through competition with a biotinylated CPA epitope-mimicking cyclic peptide (A2-biotin, ACNWWDLTLC-GGGSK (Biotin)-NH₂), anchored to a streptavidin-coated microtiter plate, for antibody binding. The ULISA platform offers ultrasensitive detection of CPA (limit of detection of 1.3 pg mL^-1 and IC₅₀ value of 15 pg mL^-1), and no cross-reactivity was observed with other coproduced mycotoxins. These results substantially outperformed the analytical features of conventional heterogeneous immunoassays based on enzymatic detection. Additionally, the use of advanced computational tools, such as MOE and Alphafold AI, proved advantageous in elucidating the molecular interactions between the antibody and the epitope, providing insights that enhance the rational design of immunoassays. The proposed ULISA was applied to detect CPA in spiked maize samples, and the results were validated by high-performance liquid chromatography coupled to a tandem mass spectrometry detector (HPLC-MS/MS).